Forebrain and hindbrain afferents projecting to the interpeduncular nucleus (IPN) have been demonstrated in male rats by retrograde transport of fluorescent dye "fast blue" microinjected into IPN followed by intraventricular colchicine 48 hrs prior to perfusion. The most intensely labeled cells projecting to IPN were concentrated throughout the entire rostrocaudal extent of the medial habenular nuclei. Immunocytochemistry revealed small numbers of labeled medial habenular cells having substance P immunofluorescence. Additional forebrain afferents originate from septal, hypothalamic and mammillary nuclei. Hindbrain afferents projecting to IPN include the nucleus incertus, a circumscribed region overlying the dorsal tegmental nucleus. Many labeled cells in the nucleus contain leu-enkephalin-like immunofluorescence. Additional brainstem afferents include the raphe, dorsolateral tegmental nuclei and locus coeruleus. Immunocytochemical studies of IPN reveal a wide variety of endogenous peptides and biogenic amines distributed in somata and processes in a topographic and heterogeneous pattern. Substance P, cholecystokinin, vasoactive intestinal peptide, somatostatin, leu-enkephalin, dopamine-Beta-hydroxylase and serotonin were observed in IPN of male rats treated with cholchicine 48 hrs prior to perfusion. Immunofluorescent cell bodies and processes were distributed differentially among IPN subnuclei revealing new features of the organization.